Riboflavin is synthesized by a wide variety of microorganisms in amounts which greatly exceed the metabolic requirements of the organisms. Ascomycetes, such as Ashbya gossypii and Eremothecium ashybii are known for production of riboflavin by fermentation. Typically, riboflavin produced by these organisms is used as a feed additive.
Riboflavin production by other microorganisms is also known. For example, the bacteria belonging to the genera Clostridium and Bacillus, as well as various genera of yeast, including Candida, Saccharomyces, Hansenula, and Pichia are known for riboflavin production. More specifically, for example, U.S. Pat. No. 3,433,707 (1969) to Matsubayashi, et al. describes the production of riboflavin by three species of Pichia yeast. Yields of riboflavin of between 10.5 mg/l and 51 mg/l in 12 days were reported. Riboflavin over-production by Ashbya gossypii of 6.42 g/l has been reported by Szczesniak et al. (1973) as discussed in Perlman, Primary Products of Metabolism. 2 Econ. Microbiology, at 312 (1978).
Commonly assigned United States patent application Ser. No. 061,811,234 filed Dec. 20, 1985, reports the development of strains of Candida famata having increased riboflavin production. The strains of C. famata discussed in Ser. No. 061,811,234 produced 5 grams of riboflavin per liter in six days of fermentation.
Study of the metabolic requirements of riboflavin producing yeast have reported a riboflavin production sensitivity to concentrations of iron. For example, Straube, et al., The Influence of Iron Concentration and Temperature on Growth and Riboflavin Overproduction of Candida guilliermondii, Biotechnology and Bioengineering Symposium No. 4, 225-231 (1973) reports that iron concentrations of 10.sup.-5 M almost completely inhibit the production of riboflavin and that riboflavin production is approximately inversely proportional to the iron concentration. Straube, et al. also found that the presence of cobalt can reverse the riboflavin production inhibition effect of iron. At cobalt concentrations of 10.sup.-4 M and iron concentration of 10.sup.-5 M, the same amount of riboflavin was produced as when no cobalt was present and iron was limited to 10.sup.-7 M.
Other references discuss iron sensitivity in riboflavin producing microorganisms and, in particular, that iron sensitivity can be partially overcome by addition of cobalt, zinc, or chelating agents to the fermentation medium. Chopde, et al., Factors Influencinq Riboflavin Synthesis by Cytophaga Hutchinsonii, Indian J. of Microbiology, v. 20 n.2 (1980); Schlee, et al., Physiology and Biochemistry of Riboflavin Formationashybii, Die Pharmazie, No. 12, (1984).
While iron inhibits riboflavin production, it has also been reported that increased amounts of iron stimulate cell growth. Therefore, yeast having decreased sensitivity to iron inhibition of flavinogenesis should produce high amounts of riboflavin because higher cell densities can be achieved by increasing iron concentrations.
Strains of riboflavin producing microorganisms having decreased sensitivity of riboflavin production to the presence of iron have been reported. Russian Patent No. SU 330189 (abstract); German Patent No. DD 108767 (abstract).
Accordingly, there is a need for microorganisms having improved levels of riboflavin production. There is a further need for strains of flavinogenic microorganisms having resistance to iron inhibition of riboflavin production. Additionally, there is a need for improved methods for selecting riboflavin over-producing microorganisms. There is also a need for fermentation media supporting increased cell growth and flavinogenesis. The microorganisms of the present invention produce riboflavin in amounts which are greatly in excess of wild type strains of flavinogenic microorganisms and which are highly improved over yields of known developed strains.